Abstract 2446 Phosphate Sensing and Mitochondrial Membrane Potential Regulation in Mammalian Cells

Mitochondria sustain the mitochondrial membrane potential (MMP), an essential electrochemical gradient across their inner membrane that is vital for various processes, including ATP synthesis.Reduced MMP levels have been associated with aging and cell death, while an increase in MMP correlates with extended lifespan in model organisms like worms and flies.However, the mechanisms controlling MMP remain poorly understood.Recent studies from our lab revealed that depleting extracellular phosphate (Pi) or disrupting Pi regulatory signaling induces mitochondrial hyperpolarization in yeast through both OxPhos-dependent and independent pathways.Similarly, Pi depletion in mammalian cells leads to increased MMP, although the underlying mechanisms and signaling networks involved in Pi sensing are not fully understood.To address this, we aimed to unravel the mechanisms by which Pi starvation influences MMP in mammalian cells.We hypothesized that Pi depletion activates signaling cascades that enhance MMP through either increased respiration or breakdown of ATP.To test this, we cultured A375 cells under normal and Pi-starved conditions for three days, then assessed MMP using Mitotracker Red and Green fluorescence ratios.We also measured intracellular Pi and ATP levels using colorimetric and cell-based luminescent assays, respectively.We also performed respirometry assays to evaluate whether elevated OxPhos activity accounted for the observed MMP increase.Additionally, we carried out a timecourse transcriptomics analysis and a CRISPR screen targeting kinases and mitochondrial genes to further understand the signaling pathways involved in both MMP regulation and Pi response.Our results demonstrated a significant increase in MMP after three days of Pi starvation, accompanied by elevated intracellular Pi levels and greater ATP depletion compared to control cells.Interestingly, mitochondrial respiration was notably reduced in Pi-starved cells.These findings suggest that the MMP rise under Pi-starvation conditions may be driven not by heightened respiration but by ATP hydrolysis, potentially contributing to intracellular Pi accumulation.This mirrors similar observations in yeast, implying a conserved Pi regulatory mechanism across species that remains to be elucidated.While the specific genes and pathways responsible for MMP modulation under Pi starvation are still being investigated, ongoing transcriptomic analyses and CRISPR screens are expected to shed light on these regulators.Future work will focus on identifying key players in MMP regulation and exploring their implications for cellular health.Overall, understanding how Pi influences MMP may provide therapeutic opportunities for conditions linked to reduced MMP, such as neurodegenerative diseases and aging, and expand our understanding of Pi sensing and regulation in mammals.